B cells sense and manipulate the lateral organization of numerous cell surface receptors in order to facilitate their functional roles within the immune system. Receptor clustering as a physical mode of signal initiation is ubiquitous in B cells, yet the corresponding general mechanisms by which clustering is sensed and controlled by intracellular components are unknown. A mechanistic understanding of clustering-mediated B cell signaling events is essential because they play important roles in immune function, defects lead to diseases such as cancer and immunodeficiency, and widely used immunotherapeutic drugs exploit these mechanisms. The goal of this work is to develop a framework that describes how receptor organization is tied to receptor functions for the class of B cell surface receptors that partition with ordered membrane domains. The working hypothesis is that ordered domain stabilization provides a fundamental paradigm for the initiation and regulation of diverse B cell signaling responses. The proposed research is guided by a predictive model of B cell receptor (BCR) signaling developed in the previous funding cycle and experimentally tests predictions of this model extended beyond BCR signaling alone. Guided by extensive preliminary data, three specific aims will be pursued: 1) Establish a generalized mechanism of signaling activated by clustering B cell surface proteins, 2) Modulate B cell membrane organization and signaling through optogenetic control of scaffolding elements, and 3) Identify immunomodulatory roles facilitated by membrane domains in BCR signaling. The first aim will establish a general sensing mechanism that describes signals initiated via clustering of more than 15 distinct B cell surface proteins that are reported in the literature to partition with ordered domains. The second aim will define how scaffolding elements template functional membrane organization that spans plasma membrane leaflets and the contribution of this effect to ligand-independent signaling. The third aim will identify and isolate the roles that phase-like membrane domains play in downstream cellular decision-making by modulating signals initiated through the BCR. All aims use quantitative super-resolution fluorescence localization microscopy techniques with the sensitivity to detect subtle domain-mediated interactions in chemically fixed and live cells. The proposed work is innovative because it applies predictive models of membrane organization and exploits recent advances in super-resolution imaging, biosensor technology, and optogenetics. A broadly applicable mechanistic model for B cell signaling will drive future advances in basic B cell biology, elucidate the mechanisms underlying the efficacy of several widely used B cell-targeted drugs, and provide new approaches for the treatment of immune diseases.